Relationship between in vitro susceptibility test results and treatment outcomes for gram-positive mastitis pathogens following treatment with cephapirin sodium

The selection of antimicrobial agents for the treatment of mastitis has often been based on results of in vitro susceptibility testing. However, the results of in vitro susceptibility tests have been shown to be poor predictors of treatment outcomes. The objective of this study was to determine if an association existed between results of antimicrobial susceptibility tests and outcomes of mastitis caused by gram-positive pathogens recovered from quarters that received treatment with cephapirin sodium. Mastitis pathogens were obtained from a multi-site clinical trial that evaluated the benefits of using an on-farm culturing system. Target pathogens (n = 187) comprised coagulase-negative staphylocci (65%), Streptococcus spp. (14%), other pathogens (12%), and Staphylococcus aureus (11%), which were recovered from quarters that received treatment using cephapirin sodium. The antimicrobial susceptibility profile to cephapirin was determined using the broth micro-dilution technique. The overall bacteriological cure rate achieved by cephapirin treatment was 82%. Bacteriological outcomes (cure or treatment failure) were not associated with pathogen type. A recurrent case of mastitis was observed in 10 quarters classified as cures and 3 quarters classified as treatment failures. Recurrence of mastitis was not associated with bacteriological outcomes or susceptibility test results. In vitro susceptibility to cephapirin was exhibited by 94.8 and 91.2% of pathogens recovered from quarters classified as cures and treatment failures, respectively. Bacteriological outcomes of mastitis treated using cephapirin were not associated with in vitro susceptibility test results or in vitro minimum inhibitory concentration values. In this population, there was an 82% probability of treatment success when the isolate was susceptible but only a 27% probability of treatment failure when the isolate was resistant. Based on this research, results of in vitro susceptibility tests should not be used as the primary guide for treatment decisions regarding intramammary cephapirin sodium.     

A survey of mastitis pathogens including antimicrobial susceptibility in southeastern Australian dairy herds

The objectives for this study were to (1) describe the pathogen profile in quarters from cows with clinical mastitis and in cows with subclinical mastitis in southeastern Australia; and (2) describe antimicrobial susceptibility among isolated pathogens. As a secondary objective, we aimed to compare antimicrobial resistance prevalence in pathogens isolated from clinical and subclinical mastitis samples. A convenience sample of dairy herds (n = 65) from 4 regions in southeastern Australia (Gippsland, Northern Victoria, Tasmania, Western Victoria) were invited to submit milk samples from cows with clinical and subclinical mastitis over a 14-mo period (January 2011 to March 2012). Farmers were instructed to collect aseptic quarter milk samples from the first 10 cases of clinical mastitis for each month of the study. In addition, farmers submitted composite milk samples from cows with subclinical mastitis at 1 or 2 sampling occasions during the study period. Aerobic culture and biochemical tests were used to identify isolates. Isolates were classified as susceptible, intermediate, or resistant to a panel of antimicrobial agents based on the zone of growth inhibition around antimicrobial-impregnated disks, with antimicrobial resistance (AMR) classified as nonsusceptibility by combining intermediate and resistant groups into a single category. Generalized linear mixed models were used to compare the prevalence of AMR between clinical and subclinical mastitis isolates. For clinical mastitis samples (n = 3,044), 472 samples (15.5%) were excluded for contamination. Of the remaining samples (n = 2,572), the most common results were Streptococcus uberis (39.2%), no growth (27.5%), Staphylococcus aureus (10.6%), Escherichia coli (8.4%), and Streptococcus dysgalactiae (6.4%). For subclinical mastitis samples (n = 1,072), 425 (39.6%) were excluded due to contamination. Of the remaining samples (n = 647), the most common results were no growth (29.1%), Staph. aureus (29.1%), and Strep. uberis (21.6%). The prevalence of AMR among common isolates was low for the majority of antimicrobial agents. Exploratory analysis found that the probability of Staph. aureus demonstrating resistance to penicillin was 5.16 times higher (95% confidence interval: 1.68, 15.88) in subclinical isolates relative to clinical Staph. aureus isolates. A similar association was observed for amoxicillin with subclinical Staph. aureus isolates being 4.70 times (95% confidence interval: 1.49, 14.75) more likely to be resistant than clinical Staph. aureus isolates. We concluded that the most common bacteria causing clinical mastitis in dairy herds in Australia is likely to be Strep. uberis, whereas Staph. aureus is likely to be the most common cause of subclinical mastitis. Despite decades of antimicrobial use to control these organisms, AMR appears to be uncommon.     

Prevalence of mastitis pathogens and their resistance against antimicrobial agents in dairy cows in Brandenburg, Germany

The primary objective of this study was to determine management practices concerning mastitis in Brandenburg, Germany, the prevalence of mastitis pathogens in dairy cows, and their resistance to selected antimicrobial agents. A further objective was to study the potential effect of parity and stage of lactation on the resistance of Staphylococcus aureus isolates against ampicillin. Milk samples for microbiological culture were collected from 4 groups of clinically healthy cows (first lactation, >1 lactation, >50 d in milk, and >250 d in milk; 8 cows/group) in 80 dairy herds. Resistance of gram-positive pathogens against 6 antimicrobial agents was tested using the broth microdilution method. Mastitis pathogens were isolated from 26.4% of the milk samples. Coagulase-negative staphylococci (CNS, 9.1% of quarters) and Corynebacterium bovis (7.3%) were the pathogens most frequently isolated. Among the major pathogens, Staph. aureus (5.7%) and Streptococcus uberis (1.0%) had the highest prevalence. Streptococcus agalactiae was isolated in samples from 29% of the herds. Although the prevalence of most pathogens was higher in older cows, the prevalence of CNS was higher in primiparous cows. Results of the mastitis control questionnaire showed that cows with clinical mastitis were transferred to a sick cow pen in 70% of the herds. Cephalosporins were the drug of first choice for treatment of clinical mastitis cases followed by fixed combinations of antimicrobial agents, β-lactamase-resistant penicillins, and penicillin. Most farmers treated cows 3 to 4 times per case. Cloxacillin, alone or in combination, and penicillin were most often used for dry-cow therapy. Antimicrobial resistance of the pathogens was within the range of other reports. Resistance of Staph. aureus to ampicillin increased significantly during the first lactation. Further research is required to determine the factors that lead to the selection of Staph. aureus strains that are resistant to ampicillin during the first lactation.     

Comparison of antibiotic resistance of udder pathogens in dairy cows kept on organic and on conventional farms

There has been a rapid rise in the emergence of multi-drug-resistant pathogens in the past 10 to 15 yr and some bacteria are now resistant to most antimicrobial agents. Antibiotic use is very restricted on Swiss organic dairy farms, and a purely prophylactic use, such as for dry cow mastitis prevention, is forbidden. A low prevalence of antibiotic resistance in organic farms can be expected compared with conventional farms because the bacteria are infrequently or not exposed to antibiotics. The occurrence of antibiotic resistance was compared between mastitis pathogens (Staphylococcus aureus, nonaureus staphylococci, Streptococcus dysgalactiae, Streptococcus uberis) from farms with organic and conventional dairy production. Clear differences in the percentage of antibiotic resistance were mainly species-related, but did not differ significantly between isolates from cows kept on organic and conventional farms, except for Streptococcus uberis, which exhibited significantly more single resistances (compared with no resistance) when isolated from cows kept on organic farms (6/10 isolates) than on conventional farms (0/5 isolates). Different percentages were found (albeit not statistically significant) in resistance to ceftiofur, erythromycin, clindamycin, enrofloxacin, chloramphenicol, penicillin, oxacillin, gentamicin, tetracycline, and quinupristin-dalfopristin, but, importantly, none of the strains was resistant to amoxicillin-clavulanic acid or vancomycin. Multidrug resistance was rarely encountered. The frequency of antibiotic resistance in organic farms, in which the use of antibiotics must be very restricted, was not different from conventional farms, and was contrary to expectation. The antibiotic resistance status needs to be monitored in organic farms as well as conventional farms and production factors related to the absence of reduced antibiotic resistance in organic farms need to be evaluated.     

Prevalence and antimicrobial susceptibility of gram-negative bacteria isolated from bovine mastitis between 2003 and 2008 in Korea

The objective of this study was to assess trends in the prevalence and distribution of gram-negative bacteria isolated from bovine mastitis and their antimicrobial susceptibilities during a 6-yr period between 2003 and 2008 in Korea. Escherichia coli, Pseudomonas fluorescens, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter lwoffi/junii, Pseudomonas aeruginosa, and Serratia marcescens were the most commonly observed pathogens during this period. Generally, gram-negative bacteria showed low susceptibilities to most of the antimicrobials tested in this study, except amikacin and gentamicin. Although these 2 aminoglycosides were broadly active against gram-negative bacteria, less than half of those bacteria showed susceptibilities to streptomycin. The β-lactams, except piperacillin, had the lowest activity among antimicrobials tested in this study. Susceptibilities to chloramphenicol and trimethoprim were fairy high in all genera of gram-negative bacteria, except Acinetobacter spp. and Pseudomonas spp., whereas relatively high resistance to tetracycline was observed uniformly among gram-negative bacteria. There was no significant change in the prevalence of bacterial and the proportion of antimicrobial resistance among gram-negative bacteria isolates during a 6-yr period.     

Treatment practices and quantification of antimicrobial drug usage in conventional and organic dairy farms in Wisconsin

The objective of this study was to develop a method to quantify antimicrobial drug usage and treatment practices on conventional and organic dairy farms that had been recruited to represent a broad spectrum of potential exposure to antimicrobial drugs. Data on disease prevalence and treatment practices of organic (n = 20) and conventional (n = 20) farms were obtained during a farm visit using a survey instrument. A standardized estimate of antimicrobial drug usage was developed using a defined daily dose (DDD) of selected compounds. Density of antimicrobial drug usage was expressed as the number of DDD per adult cow per year. Differences in prevalence and management of selected diseases between conventional and organic farms were identified. The overall estimated prevalence of selected diseases was greater for conventional farms compared with organic farms. Organic farmers reported use of a variety of nonantimicrobial compounds for treatment and prevention of disease. Conventional farmers reported that penicillin was the compound most commonly used for dry cow therapy and cephapirin was most commonly used for treatment of clinical mastitis. On conventional farms, the estimated overall exposure to antimicrobial drugs was 5.43 DDD per cow per year composed of 3.58 and 1.85 DDD of intramammary and parenteral antimicrobial drugs, respectively. Of total intramammary antimicrobial drug usage, treatment of clinical mastitis contributed 2.02 DDD compared with 1.56 DDD attributed to the use of dry cow therapy. Of total parenteral treatments, the distribution of exposure was 0.52 (dry cow therapy), 1.43 (clinical mastitis treatment), 0.39 (treatment of foot disease), 0.14 (treatment of respiratory disease), and 0.32 (treatment of metritis) DDD. For treatments of foot infections (0.33 DDD), respiratory infections (0.07 DDD), and metritis (0.19 DDD), the mean density of ceftiofur usage was significantly greater compared with other compounds.     

Negatively controlled,randomized clinical trial comparing different antimicrobial interventions for treatment of clinical mastitis caused by gram-positive pathogens

The general objective of this study was to evaluate the effect of 3 intramammary antibiotic interventions using 2 commercially available antibiotics with narrow- or broad-spectrum activity on cure rates of clinical mastitis (CM) caused by gram-positive bacteria. We also compared the efficacy of treatment protocols, including a negative control, on outcomes at the cow and mammary quarter level. Before the onset of the study, 5,987 animals more than 12 mo old were randomly preassigned to 1 of 4 protocols in the event of gram-positive CM (except for Staphylococcus aureus and Trueperella pyogenes) during lactation: 3 infusions with 62.5 mg of amoxicillin performed 12 h apart (AMOX-L); 5 infusions once a day with 62.5 mg of amoxicillin (AMOX-EL); 5 infusions once a day with 125 mg of ceftiofur hydrochloride (CEFT-L); or negative control, no treatment performed until 5 d after diagnosis (NEG-CTR). Randomization was performed to preassign 90% of cows to one of the antibiotic protocols (30% in each group) and 10% to the negative control. A total of 696 quarter cases of CM met the inclusion criteria and were evaluated in the study. Quarter-level outcomes were assessed based on 5 milk samples collected up to 14 ± 3 d following enrollment (i.e., first day of treatment), whereas variables at the cow level [composite somatic cell count (SCC), milk production, and survival in the herd] were assessed up to 90 d after CM diagnosis. Streptococcus uberis, followed by Streptococcus dysgalactiae, were the main causes of gram-positive CM. Overall, clinical cure was higher for CEFT-L than for AMOX-EL, and no difference was observed between CEFT-L and AMOX-L. Likewise, no significant differences were detected on overall bacteriological cure, although some treatment effects were observed at the species level. Compared with antibiotic-treated groups, quarters assigned to NEG-CTR had higher counts of colony-forming units (cfu), 16S rRNA gene copy numbers, and Streptococcus relative abundance (RA) until d 5 after enrollment. Quarters treated with AMOX-L had higher cfu counts on d 5, 8, and 14 after enrollment compared with the other antibiotic protocols. In addition, the RA of Streptococcus spp. was higher on d 14 after enrollment for AMOX-treated quarters compared with the CEFT-L group. Linear score of SCC was higher for AMOX-treated cows than for CEFT-L in the first test day after CM. However, cows assigned to AMOX-L had higher milk production than those submitted to the AMOX-EL and CEFT-L protocols. In conclusion, the 2-d protocol with 3 intramammary infusions of amoxicillin (narrow-spectrum antimicrobial) had similar overall clinical and bacteriological cures as 5 administrations (once a day) with ceftiofur hydrochloride (wide spectrum). No significant difference was observed on CM recurrence and cow survival. However, quarters treated with 5-d protocols were more effective at reducing milk cfu counts than quarters in the AMOX-L protocol. In addition, lower Streptococcus spp. RA was observed in ceftiofur-treated quarters compared with the amoxicillin protocols at d 14 after CM diagnosis. Based on results of microbiome and bacterial load (quantitative PCR and cfu count) up to 5 d after CM diagnosis, antibiotic use remains an indispensable strategy for treatment of CM caused by gram-positive bacteria.     

Evaluating the in vitro susceptibility of bovine mastitis pathogens to a combination of kanamycin and cefalexin: Recommendations for a disk diffusion test

Cows suffering from bovine mastitis have markedly reduced milk production because of inflammation within the udder subsequent to infection and damage from bacterial toxins. Antibiotic treatment is commonly used as a preventative and therapeutic measure for bovine mastitis. The most common pathogens include Staphylococcus aureus, various streptococci (Streptococcus dysgalactiae, Streptococcus uberis), and coliforms (Escherichia coli), which can be contracted from other infected cows or from the environment. A combination of kanamycin and cefalexin (1:1.5 wt/wt) is currently used therapeutically in Europe for the treatment of bovine mastitis, although standardized methods for the in vitro determination of the susceptibility of target pathogens have not been developed. This study evaluates the appropriate broth microdilution testing criteria for kanamycin and cefalexin administered in combination and reports the development of a disk diffusion test. At a ratio of kanamycin:cefalexin relevant to that observed in milk postadministration (10:1 wt/wt), the minimum inhibitory concentrations were determined against 307 isolates of target mastitis pathogens (staphylococci, streptococci, and E. coli). Based on achievable concentrations in milk and the resulting distribution of minimum inhibitory concentrations, preliminary broth breakpoints for kanamycin/cefalexin (10:1 fixed ratio) of ≤8/0.8 μg/mL susceptible, 16/1.6 μg/mL intermediate, and ≥32/3.2 μg/mL resistant were applied to evaluated staphylococci, streptococci, and E. coli. Parallel testing by disk diffusion and resulting error-rate bounded analysis using a combined disk concentration of 30 μg of kanamycin and 15 μg of cefalexin resulted in the establishment of preliminary disk interpretive breakpoints of ≥20 mm susceptible, 18 to 19 mm intermediate, and ≤17 mm resistant for staphylococci, streptococci (Strep. uberis and Strep. dysgalactiae only), and E. coli.     

Phenotypic and genotypic antimicrobial susceptibility pattern of Streptococcus spp. isolated from cases of clinical mastitis in dairy cattle in Poland

Mastitis of dairy cattle is one of the most frequently diagnosed diseases worldwide. The main etiological agents of mastitis are bacteria of the genus Streptococcus spp., in which several antibiotic resistance mechanisms have been identified. However, detailed studies addressing this problem have not been conducted in northeastern Poland. Therefore, the aim of our study was to analyze, on phenotypic and genotypic levels, the antibiotic resistance pattern of Streptococcus spp. isolated from clinical cases of mastitis from dairy cattle in this region of Poland. The research was conducted using 135 strains of Streptococcus (Streptococcus uberis, n = 53; Streptococcus dysgalactiae, n = 41; Streptococcus agalactiae, n = 27; other streptococci, n = 14). The investigation of the antimicrobial susceptibility to 8 active substances applied in therapy in the analyzed region, as well as a selected bacteriocin (nisin), was performed using the minimum inhibitory concentration method. The presence of selected resistance genes (n = 14) was determined via PCR. We also investigated the correlation between the presence of resistance genes and the antimicrobial susceptibility of the examined strains in vitro. The highest observed resistance of Streptococcus spp. was toward gentamicin, kanamycin, and tetracycline, whereas the highest susceptibility occurred toward penicillin, enrofloxacin, and marbofloxacin. Additionally, the tested bacteriocin showed high efficacy. The presence of 13 analyzed resistance genes was observed in the examined strains [gene mef(A) was not detected]. In most strains, at least one resistance gene, mainly responsible for resistance to tetracyclines [tet(M), tet(K), tet(L)], was observed. However, a relationship between the presence of a given resistance gene and antimicrobial susceptibility on the phenotypic level was not always observed.     

Relationship between milk cathelicidin abundance and microbiologic culture in clinical mastitis

The availability of reliable tools to enable the sensitive and specific detection of mastitis in dairy cows can assist in developing control strategies and promote the more rational use of antibiotics. We have developed a milk cathelicidin ELISA that shows high sensitivity and specificity for dairy cow mastitis, based on latent class analysis. In this study, we investigated the effect of microbial agents on cathelicidin abundance in the milk of cows with clinical mastitis. We subjected 535 quarter milk samples (435 from quarters showing signs of clinical mastitis and 100 from healthy quarters as a control) to milk cathelicidin ELISA, somatic cell count (SCC), and microbiologic culture. Of the 435 clinical mastitis samples, 431 (99.08%) were positive for cathelicidin, 424 (97.47%) had SCC >200,000 cells/mL, and 376 (86.44%) were culture-positive. Of the 59 culture-negative samples, 58 (98.30%) were positive for cathelicidin and 55 (93.22%) had SCC >200,000 cells/mL. The abundance of cathelicidin and the extent of SCC increase depended on the causative agent: Streptococcus agalactiae and coagulase-negative staphylococci showed the highest and lowest changes, respectively. We also observed differences in behavior between the 2 markers depending on the pathogen: Streptococcus agalactiae induced the highest cathelicidin abundance, and Serratia spp. induced the highest SCC. Nevertheless, the different ability of microorganisms to induce cathelicidin release in milk did not compromise its value as a mastitis marker, given its higher sensitivity compared to SCC or microbiologic culture. All 100 negative control samples (collected from healthy quarters with SCC <100,000 cells/mL and culture-negative) were also negative for cathelicidin, corresponding to 100% specificity in the evaluated sample cohort. This study confirmed the value of the milk cathelicidin ELISA for detecting bovine mastitis, and highlighted the influence of mastitis-causing microorganisms on cathelicidin abundance. This influence did not compromise diagnostic performance; instead, it may have better reflected disease severity and evolution than SCC.     

Trends in antibacterial susceptibility of mastitis pathogens during a seven-year period

Milk samples collected from dairy cattle suspected of having mastitis were submitted to the Microbiology Laboratory of the Animal Health Diagnostic Laboratory, Michigan State University, for bacteriologic culture. A total of 2778 isolates, from the years 1994 to 2000, were isolated, identified, and subjected to in vitro antimicrobial susceptibility testing using the disk diffusion method, in accordance with National Committee on Clinical Laboratory Standards (NCCLS) standards. Isolates included in this study were Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus agalactiae, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Serratia marcesens, and Pseudomonas aeruginosa. The proportion of bacterial isolates determined to be susceptible did not change during the 7-yr period for the majority of bacterial-antibacterial interactions tested. However, analysis for linear trend in proportions determined that there were increases in the proportion of S. aureus isolates that were susceptible to ampicillin, penicillin, and erythromycin. For Strep. uberis, increases in the proportion of susceptible isolates occurred for oxacillin, sulfa-trimethoprim, gentamicin, and pirlimycin, and a decrease in the proportion of susceptible isolates occurred with penicillin. For Strep. dysgalactiae, increases in the proportion of susceptible isolates occurred with erythromycin, gentamicin, sulfa-trimethoprim, and tetracycline. For Strep. agalactiae, increases in the proportion of susceptible isolates occurred with sulfa-trimethoprim. Among E. coli isolates, there was an increase in the proportion that were susceptible to ampicillin and cephalothin. Among K pneumoniae isolates, there was an increase in the proportion that were susceptible to ceftiofur. Overall, there was no indication of increased resistance of mastitis isolates to antibacterials that are commonly used in dairy cattle.     

Effects of clinical mastitis caused by gram-positive and gram-negative bacteria and other organisms on the probability of conception in New York State Holstein dairy cows

The objective of this study was to estimate the effects of different types of clinical mastitis (CM) on the probability of conception in New York State Holstein cows. Data were available on 55,372 artificial inseminations (AI) in 23,695 lactations from 14,148 cows in 7 herds. We used generalized linear mixed models to model whether or not a cow conceived after a particular AI. Independent variables included AI number (first, second, third, fourth), parity, season when AI occurred, farm, type of CM (due to gram-positive bacteria, gram-negative bacteria, or other organisms) in the 6 wk before and after an AI, and occurrence of other diseases. Older cows were less likely to conceive. Inseminations occurring in the summer were least likely to be successful. Retained placenta decreased the probability of conception. Conception was also less likely with each successive AI. The probability of conception associated with the first AI was 0.29. The probability of conception decreased to 0.26, 0.25, and 0.24 for the second, third, and fourth AI, respectively. Clinical mastitis occurring any time between 14 d before until 35 d after an AI was associated with a lower probability of conception; the greatest effect was an 80% reduction associated with gram-negative CM occurring in the week after AI. In general, CM due to gram-negative bacteria had a more detrimental effect on probability of conception than did CM caused by gram-positive bacteria or other organisms. Furthermore, CM had more effect on probability of conception immediately around the time of AI. Additional information about CM (i.e., its timing with respect to AI, and whether the causative agent is gram-positive or gram-negative bacteria, or other organisms) is valuable to dairy personnel in determining why some cows are unable to conceive in a timely manner. These findings are also beneficial for the management of mastitic cows (especially those with gram-negative CM) when mastitis occurs close to AI.     

Differences between Staphylococcus aureus lineages isolated from ovine and caprine mastitis but not between isolates from clinical or subclinical mastitis

Staphylococcus aureus is an important mastitis pathogen, causing both clinical mastitis (CM) and subclinical mastitis (SCM) in small ruminants. In general, CM has a low incidence in sheep and goats but can be very severe and costly. In contrast, subclinical mastitis (SCM) is common but is associated with less cost. For both sheep and goats, S. aureus is the main cause of CM and is associated with SCM cases with a high SCC. Recently, specific lineages of S. aureus have been identified that are associated with CM rather than SCM in dairy cows. It is unknown whether specific S. aureus lineages are associated with CM in goats and sheep. The aim of this study was to compare the clonal complex (CC), staphylococcal protein A (spa) type, leukocidin lukM-lukF' presence, and potential to produce LukMF' in vitro between CM and SCM S. aureus mastitis isolates obtained from sheep and goats. Differences between isolates from different host species were also compared. Ovine (CM, n = 12; SCM, n = 29) and caprine (CM, n = 14; SCM, n = 30) isolates were obtained from 8 sheep flocks and 8 goat herds in the Netherlands. Overall, the isolates belonged to CC133 (85%), CC398 (7%), CC425 (5%), and CC45 (2%). Seventeen spa types were found, including 6 novel types; the predominant types were t2678 (34%), t544 (18%), and t3583 (18%). Although CC133 was dominant among both sheep and goat isolates, spa type CC133/t2678 was associated with ovine isolates, whereas CC133/t544 and CC133/t3583 were found mostly in goats. The presence of lukM-lukF' among the S. aureus isolates was high (87%), especially in CC133 (96%) and CC425 (100%), but the genes were absent in CC45 and CC398. In vitro-cultured lukM-lukF'-positive isolates produced LukM (71 out of 74 positive isolates tested) in the range of 0.4 to 5.0 µg/mL. Interestingly, the goat-associated lineages CC133/t544 and CC133/t3583 produced more LukM in vitro than the sheep-associated CC133/t2678. We found no difference in LukMF' production potential between CM and SCM isolates. In sheep as well as in goats, no association was found between genotype and CM or SCM, demonstrating that the same lineages of S. aureus are responsible for both CM and SCM. These results suggest that subclinically infected animals in a herd or flock likely act as the reservoir of S. aureus causing CM. This highlights the importance of early identification and control of SCM and suggests that controlling SCM within a herd is an effective intervention to prevent CM in small ruminants.     

Invited review: Low milk somatic cell count and susceptibility to mastitis

An enduring controversy exists about low milk cell counts and susceptibility to mastitis. The concentration of milk leukocytes, or somatic cell count (SCC), is a well-established direct indicator of mammary gland inflammation that is highly correlated with the presence of a mammary infection. The SCC is also used as a trait for the selection of dairy ruminants less prone to mastitis. As selection programs favor animals with less SCC, and as milk cells contribute to the defense of the mammary gland, the idea that susceptibility to mastitis could possibly be increased in the long term has been put forward and is still widely debated. Epidemiological and experimental studies aimed at relating SCC to susceptibility to mastitis have yielded results that seem contradictory at first sight. Nevertheless, by taking into account the immunobiology of milk and mammary tissue cells and their role in the defense against infection, along with recent studies on SCC-based divergent selection of animals, the issue can be settled. Apparent SCC-linked susceptibility to mastitis is a phenotypic trait that may be linked to immunomodulation but not to selection.     

Determinants of antimicrobial treatment for udder health in Danish dairy cattle herds

Societal pressure to limit the use of antibiotics in livestock production systems, including dairy cattle systems, is consistently increasing. To motivate farmers to reduce antibiotic usage, it is important to understand the factors that determine whether a cow will be treated with antibiotics or not. If farmers' usual practices regarding antibiotic treatments are taken into account, they may be motivated to adopt control measures that can facilitate prudent use of antibiotics and are at the same time cost-effective. In this study, we analyzed database recordings of milk yield and somatic cell count from the routine milk recording scheme, clinical registrations of mastitis and PCR results, and cow factors such as days in milk and parity in relation to antibiotic treatments for 518 dairy herds in Denmark. Farm-wise logistic regressions were used to predict antimicrobial treatment based on these factors. The resulting regression coefficients of 422 herds were further analyzed by principal component analysis and clustering to determine the driving predictors for treatment in different groups of farms. The results showed that determinants that were most important for predicting antibiotic treatments vary from one farm to another. Health indicators such as PCR or somatic cell count were most indicative for treatment on some farms, whereas other groups seemed to depend more on production factors (milk yield) or later culling of the cows. This shows that farmers behave differently and differences can be identified in register data. This information can be considered when developing cost-effective herd-specific control measures of mastitis to promote prudent use of antibiotics in Danish dairy cattle farms.     

Short communication: Molecular characteristics,antimicrobial susceptibility,and pathogenicity of clinical Nocardia cyriacigeorgica isolates from an outbreak of bovine mastitis

The occurrence of nocardial mastitis, mostly in the context of outbreaks, has been reported in many countries. However, there is a paucity of reports regarding detailed characterization of Nocardia cyriacigeorgica from bovine mastitis. Thus, herein we report characteristics, antimicrobial susceptibility patterns, molecular identification, and pathogenicity of N. cyriacigeorgica isolated from an outbreak of clinical mastitis in a dairy herd in northern China. A total of 182 (80.2%) lactating cows had clinical mastitis with severe inflammation and firmness of the udder, reduced milk production, and anorexia, with no apparent clinical response to common antibiotics. Out of 22 mastitic milk samples submitted to our laboratory, 12 N. cyriacigeorgica were isolated and characterized using standard microbiological analysis, 16S rRNA gene sequencing, random amplified polymorphic DNA PCR analysis, biochemical assays, and antibiotic susceptibility testing. Additionally, in vivo experiments were done to determine pathogenicity of these clinical mastitis isolates. All isolates were resistant to ampicillin, amoxicillin-clavulanic acid, ciprofloxacin, minocycline, rifampicin, and aminoglycosides (type VI pattern). Additionally, intramammary inoculation of mice with N. cyriacigeorgica caused chronic inflammatory changes, including hyperemia, edema, and infiltration of lymphocytes and neutrophils, as well as hyperplasia of lymph nodules in mammary glands. Therefore, we concluded that N. cyriacigeorgica was involved in the current outbreak of mastitis. To our best knowledge, this is the first report to characterize N. cyriacigeorgica isolated from cases of bovine mastitis in China.     

Prevalence of mastitis pathogens in Ragusa, Sicily, from 2000 to 2006

The objective of this study was to report the prevalence of intramammary infections (IMI) in Ragusa, Sicily, from milk samples (n = 18,711) collected between October 2000 and June 2006 from 101 dairy herds. Milk samples were collected at 9,747 cow sampling events from 5,285 individual cows. Samples were individual quarter (92.8%) or composite samples (7.2%) from an individual cow. Logistic regression was used to examine the prevalence of IMI at the level of milk sample and at the level of cow, controlling for year and season of collection, type of sample (individual quarter or composite), and type of housing and bedding of the cow at the time of collection. Bedding and housing types were as follows, respectively (number of herd groups): bedding: none (61), organic [51 (straw, 50; sawdust, 1)], and sand (3); housing: bedded pack (37), free stalls (57), tie stalls (4), and paddock (17). Raw prevalence of cow IMI for a sample event was as follows (percentage of cow samples): no growth, 47.4%; coagulase-negative staphylococci, 22.6%; Staphylococcus aureus, 20.6%; other Streptococcus spp., 11.1%; Streptococcus agalactiae, 2.3%; coliform bacteria, 2.9%; and other organisms, 5.8%. Prevalence of IMI differed by bedding type for Staph. aureus (none, 24.5%; organic, 12.7%; sand, 12.3%) and coagulase-negative staphylococci (none, 13.1%; organic, 27.4%; sand, 26.9%) but not for Streptococcus spp. or coliform bacteria. Prevalence of Streptococcus spp. IMI differed by housing type (tie stalls, 22.2%; bedded pack, 12.8%; free stalls, 8.4%; paddock, 7.1%). Housing was not associated with the prevalence of IMI for other bacteria. Herd monthly prevalence of Staph. aureus and Streptococcus spp. IMI was associated with decreased mean milk production (Staph. aureus, −1.42 kg/d per cow, SEM 0.51; Streptococcus spp., −1.31 kg/d per cow, SEM 0.64) and increased mean linear score (Staph. aureus, 1.01 units/d per cow, SEM 0.16; Streptococcus spp., 0.59 units/d per cow, SEM 0.22). Herds (n = 11) with a mean linear score (MLS) less than 3.3 units had the lowest prevalence of Staph. aureus IMI and monthly MLS and the greatest monthly mean milk production compared with other herds grouped by MLS [MLS 3.31 to 3.99 (n = 20), MLS 4.00 to 4.46 (n = 20), MLS >4.46 (n = 17), and MLS not available (n = 33)]. Implementation of a milk quality program to control gram-positive organisms is important for Ragusa.     

Intramammary antimicrobial treatment of subclinical mastitis and cow performance later in lactation

The aim of this study was to evaluate long-term therapeutic effects of antimicrobial treatment of recently acquired subclinical mastitis (RASCM) during lactation. Quarter-level clinical mastitis (CM) follow-up, composite somatic cell counts (SCC), and cow-level milk yield later in lactation were evaluated using follow-up data from 2 previously published linked randomized field trials. The first trial randomly assigned antimicrobial treatment with any intramammary product or negative control to culture-positive quarters of cows having a first elevated composite SCC after 2 consecutive low composite SCC measurements. Untreated cows that had a second elevated composite SCC at the next measurement and were staphylococci-positive (i.e., Staphylococcus aureus or non-aureus staphylococci) were randomly assigned to treatment or control. Quarter-level CM cases were reported by the participating herd personnel, and milk yield and composite SCC data were obtained from the regular test-day recording. Frailty survival models were used to evaluate the long-term therapeutic effects of antimicrobial treatment of RASCM on quarter-level CM follow-up. Mixed linear regression models were applied to quantify the effect on milk yield and composite SCC. Data of 638 quarters from 486 cows in 38 herds were available for statistical analyses, of which 229 quarters of 175 cows received antimicrobial treatment for RASCM. Antimicrobial treatment culminated in reduced composite SCC levels later in lactation but did not result in different milk yield levels or CM follow-up compared with control cows. Antimicrobial treatment of cows with RASCM should therefore only be considered in exceptional situations given the current focus on antimicrobial usage reduction in animal husbandry.     

Efficacy of 5-day parenteral versus intramammary benzylpenicillin for treatment of clinical mastitis caused by gram-positive bacteria susceptible to penicillin in vitro

The efficacy of parenteral (intramuscular) or intramammary (IMM) benzylpenicillin treatment for clinical mastitis caused by gram-positive bacteria susceptible to penicillin in vitro was investigated. Cows with clinical mastitis in 1 udder quarter were randomly placed into 2 treatment groups. The preliminary bacteriological diagnosis of intramammary infection (IMI) was based on on-farm culturing, and the bacteriological diagnoses were later confirmed by a quantitative PCR assay. Clinical mastitis caused by gram-positive bacteria susceptible to benzylpenicillin was treated with penicillin via either the parenteral route (20 mg/kg) or IMM route (600 mg) once per day for 5 d. The outcome of the treatment was evaluated 3 to 4 wk after the onset of the treatment. The affected quarter was examined to assess the clinical cure, and milk samples were collected from the affected quarter to determine the bacteriological cure and milk N-acetyl-β-d-glucosaminidase activity. The survival and the composite milk somatic cell counts of the treated cows were followed up for 6 and 3 mo after treatment, respectively. A total of 140 cows with clinical mastitis were included in the study, 61 being treated with benzylpenicillin parenterally and 79 via the IMM route. From all quarters treated, 108 of 140 (77.1%) were cured clinically and 77 of 140 (55.0%) were cured bacteriologically. The route of treatment did not significantly affect the outcome of the treatment; 80.3% of the quarters with parenteral treatment and 74.7% of the quarters with IMM treatment showed a clinical cure, and 54.1 and 55.7% a bacteriological cure, respectively. The milk N-acetyl-β-d-glucosaminidase activity was significantly lower in the quarters with a clinical or bacteriological cure than in the quarters with no cure. The 6-mo survival and the proportion of cows with composite milk somatic cell counts <200,000/mL among the treated cows during the 3-mo follow-up period did not significantly differ between the treatment groups. In conclusion, the outcome of either parenteral or IMM benzylpenicillin treatment of clinical mastitis caused by penicillin-susceptible bacteria was similar.